Isolated nucleic acid molecules which encode a melanoma specific antigen and uses thereof

ABSTRACT

The invention involves the isolation of a nucleic acid molecule which encodes a melanoma associated antigen. Cell lines and expression vectors which include this and related sequences, as well as uses of these molecules, are described.

RELATED APPLICATION

This application is a Continuation-in-part of application Ser. No.08/479,328, filed on Jun. 7, 1995, now U.S Pat. No. 5,698,396.

FIELD OF THE INVENTION

This invention relates to methodologies for identifying molecules ofinterest. In particularly preferred embodiments, the invention relatesto the identification of molecules associated with pathologicalconditions such as cancer, (melanoma or renal cancer, e.g.), Hodgkin'sDisease, autoimmune diseases and so forth. Also a part of the inventionare the isolated molecules found as a result of the inventive method.These molecules include, inter alia, protein-containing molecules,isolated nucleic acid molecules encoding these, and antibodies whichspecifically bind to the protein-containing molecules. For convenience,the method described herein will be referred to as "serologicalfishing".

BACKGROUND AND PRIOR ART

It is fairly well established that many pathological conditions, such asinfections, cancer, autoimmune disorders, etc., are characterized by theinappropriate expression of certain molecules. These molecules thusserve as "markers" for a particular pathological or abnormal condition.Apart from their use as diagnostic "targets", i.e., materials to beidentified to diagnose these abnormal conditions, the molecules serve asreagents which can be used to generate diagnostic and/or therapeuticagents. A by no means limiting example of this is the use of cancermarkers to produce antibodies specific to a particular marker. Yetanother non-limiting example is the use of a peptide which complexeswith an MHC molecule, to generate cytolytic T cells against abnormalcells.

Preparation of such materials, of course, presupposes a source of thereagents used to generate these. Purification from cells is onelaborious, far from sure method of doing so. Another preferred method isthe isolation of nucleic acid molecules which encode a particularmarker, followed by the use of the isolated encoding molecule to expressthe desired molecule.

To date, two strategies have been employed for the detection of suchantigens, in, e.g., human tumors. These will be referred to as thegenetic approach and the biochemical approach. The genetic approach isexemplified by, e.g., dePlaen et al., Proc. Natl. Acad. Sci. USA 85:2275 (1988), incorporated by reference. In this approach, severalhundred pools of plasmids of a cDNA library obtained from a tumor aretransfected into recipient cells, such as COS cells, or intoantigen-negative variants of tumor cell lines which are tested for theexpression of the specific antigen. The biochemical approach,exemplified by, e.g., Falk et al., Nature 351: 290 (1991), and Kawakamiet al., Nature 369: 69 (1994) both of which are incorporated byreference, is based on acidic elution of peptides which have bound toMHC-I molecules of tumor cells, followed by reversed-phase highperformance liquid chromotogaphy (HPLC). Antigenic peptides areidentified after they bind to empty MHC-I molecules of mutant celllines, defective in antigen processing, and induction of specificreactions in cytolytic T-lymphocytes. These reactions include inductionof CTL proliferation, TNF release, and lysis of target cells, measurablein an MTT assay, or a ⁵¹ Cr release assay.

These two approaches to the molecular definition of antigens have thefollowing disadvantages: first, they are enormously cumbersome,time-consuming and expensive; second, they depend on the establishmentof cytolytic T cell lines (CTLs) with predefined specificity; third,their relevance in vivo for the course of the pathology or disease inquestion has not been proven, as the respective CTLs can be obtained notonly from patients with the respective disease, but also from healthyindividuals, depending on their T cell repertoire.

The problems inherent to the two known approaches for the identificationand molecular definition of antigens is best demonstrated by the factthat both methods have, so far, succeeded in defining only very few newantigens in human tumors. See, e.g., van der Bruggen et al., Science254: 1643-1647 (1991); Brichard et al., J. Exp. Med. 178: 489-495(1993); Coulie, et al., J. Exp. Med. 180: 35-42 (1994), Kawakami et al.,Proc. Natl. Acad. Sci. USA 91: 3515-3519 (1994).

It would be desirable to have available a method which can be used notonly for detection of tumor-associated antigens, but to determinemolecules associated with any abnormal or pathological condition. Such amethod would also facilitate the identification of such molecules,thereby enabling their use on the generation of, e.g., antibodies,cytolytic T cells, and so forth.

It is therefore the purpose of the present invention to develop a methodfor the simple detection and molecular characterization of antigens inhuman tissues, especially in tumor cells, which are useful in themolecular diagnosis of diseases and/or for immunotherapy and genetherapy of infectious, autoimmune and malignant diseases. The inventionis delineated in the disclosure which follows.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the principles of the approach of the invention.

FIG. 2 shows a nitrocellulose membrane with a positive clone derivedfrom the cDNA of a renal cell clear carcinoma that reacts with a 1:100dilution of the patient's serum.

FIG. 3 shows, in bar graph form, the Northern Blot analysis of cloneHOM-RCC-313 in renal cell carcinoma, normal kidney and other humantissues.

FIG. 4 shows the translated region of the gene coding for HOM-RCC-313.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

The following disclosure describes a methodology referred to asserological fishing. In it, a cell sample is taken from a subjectafflicted with a pathological condition. The cells preferably areexemplary of the pathology. For example, if the subject has melanoma,the cells are melanoma cells. If the subject is suffering from a neuraldisorder, e.g., then the cells are preferably a sample of the afflictedcells. This approach is warranted because the afflicted cells are mostprobably the best source of protein-containing molecules of interest,i.e., such molecules which are specifically associated with thepathological condition of interest.

Note that cells representative of pathological conditions are not theonly cells which may be used in the inventive method. It is veryimportant, e.g., to ascertain those cellular "markers" associated withdifferentiation and maturation of cells, for example. The example ofhematopoietic stem cells comes to mind. Similarly, the inventioncontemplates the isolation of, e.g., receptor molecules for specificligands. In effect, one can assay for the presence of any molecule ofinterest using this methodology.

The cells chosen are then used to prepare a library of complementary DNA(i.e., "cDNA"). This methodology is well known to the skilled artisan,and need not be reiterated here. It is, of course, based upon theestablished fact that if proteins are expressed by the cells, thenmessenger RNA (mRNA) must be present. These mRNA molecules are not longlived, and are unstable, so they are not practical to work with. Thestability brought to the molecules when cDNA is used is very helpful tothe method.

Once the cDNA is made, it is used to construct a vector library. Inshort, carrier vectors are treated, such as by cutting and splicing, toreceive molecules of cDNA. The choice of vector may vary, as the skilledartisan is well familiar with many such examples.

Especially preferred are virus based vectors. In the case of eukaryoticcells, retrovirus or adenovirus based vectors are preferred. Suchvectors contain all or a part of a viral genome, such as long termrepeats ("LTRs"), promoters (e.g., CMV promoters, SV40 promoter, RSVpromoter), enhancers, and so forth. When the host cell is a prokaryote,bacterial viruses, or phages, are preferred. Exemplary of such vectorsare vectors based upon, e.g., lambda phage. In any case, the vector maycomprise elements of more than one virus.

The resulting vectors are transfected or transformed into a host cell,which may be eukaryotic or prokaryotic.

Any cell normally used for transfection or transformation may be used inthe protocol. Preferred materials include strains of E. coli, CHO cellssuch as CHO-1, COS cells such as COS-7, and so forth. Similarly, yeastcells, e.g., strains of Saccharomyces, strains of Pseudomonas, such asstrains of Pseudomonas aeruginosa, Bacillus bacteria, Spodopterafrugiperda, a well known insect host cell, and so forth, may all beused.

Once the recipient cells receive the vectors, they are cultivated so asto express foreign, protein containing molecules. "Protein-containing"is used herein because, while prokaryotes express only proteins,eukaryotic cells are well known for their ability topost-translationally modify proteins, so as to produce glycoproteins,lipoproteins, e.g. It must also be borne in mind that "proteincontaining" as used herein, also encompasses peptides, such as thepeptides presented by MHC molecules.

The processes now described below take place independently of theprocess described above, and no chronological relationship between thetwo facets of the invention is intended.

In pathological conditions such as cancer and, e.g., autoimmunediseases, there is some immune reaction to molecules associated with thepathology. This reaction can include an antibody response, B cellproliferation, proliferation of specific T cell subpopulations,increases in cytokine production, and so forth. The molecules and cellsassociated with the response may be found in body fluids of a subject,such as his or her serum. The immune responders will react with themolecule of interest whether it is produced recombinantly orautologously. The problem is to find them. As the examples show, this isdone in a unique way. First, the body fluid, or other sample ofinterest, is reacted with a sample of the same host cells used fortransfection or transformation. In this first step, the host cells arenot transfected or transformed. The effect of this is to strip anyimmunogenic binding partners specific for the host cell rather than thetargeted molecule. This step is necessary because, as was pointed out,supra, the host cell may be one against which the subject has developedan immune respone at some point. This first stripping removes theseimmune components.

A second stripping step is then carried out. In this step, thepreviously stripped sample is now reacted with a sample of the same hostcell as was described supra, this time having been transfected ortransformed with the carrier vector lacking cDNA from the subject. Thereason for this second stripping step is an observation made by thepresent inventors not reported in the literature. The materials used asvectors, such as phages, viruses, etc., are useful because theynaturally infect cells. Thus, E. coli, which inhabit the lower intestineof humans, are infected with lambda phage. It had not been considered,previously, that the immune response to E. coli includes a response tothese infectious agents. Thus, applicants have surprisingly, achieved anability to remove interfering immune components to an unprecedenteddegree by carrying out the two stripping steps. As noted, the first isagainst untransfected or untransformed host cells. The second is againsthost cells transfected or transformed with a vector which does not carrycDNA, wherein the vector is immunologically equivalent to the vectorused to carry cDNA, as described supra.

It is especially preferred to carry out each of these stripping stepsusing a plurality of similar, but different procedures. The experimentswhich follow, for example, show absorption on a solid phase column, andthen absorption on nitrocellulose paper. Applicants do not wish to bebound by any theory as to why the use of two similar but differentprotocols produces the results described herein. It is to be borne inmind, hereafter, that whenever "contacting a sample" is used herein, itis not to be limited to one contact step only, but may refer to morethan one, preferably different contact protocols designed to removeinterfering binding partners from a sample under scrutiny.

It should be understood that these stripping steps may be donecompletely independently of the steps used to prepare the cDNA library.For example, if the test for an antigen is to be done at day "O", thestripping of sample may be done the day before, a week before, and soforth. One can also "bank" stripped sample from a donor or subject forfuture use.

The sample used is preferably serum, but need not be. Any sample whichcontains immunogenic binding partners may be so used.

In the next step of the method, lysed, transfected cells carrying thecDNA and expressing heterologous protein are contacted with the twicestripped sample. This sample should only contain immune componentsspecific for the heterologous protein, and should bind thereto. Thisbinding is facilitated if the cell lysates have been immobilized viacontact to, e.g., activated filter paper, a solid phase column, etc.,but this solid phase binding is not necessary, as the art will surelyrecognize that many, varied forms of assays are available foridentifying a molecule of interest.

Once the immune component binds to the target molecule, a further stepis desirably, but not necessarily, carried out. This additional stepinvolves the use of some binding partner for the first immune component,such as anti-IgG, carrying an identifiable label. The label may be adye, an enzyme, a gold particle, a radiolabel, or any of the standardlabels used in immunoassays.

Once identification is carried out, the immune components are removed,leaving the target molecule. The target molecule is then studied, usingany of the standard methodologies in the art.

The artisan will note that the methodology also results in isolation ofimmune components which bind to the molecule of interest. Thus, inanother aspect of the invention one can isolate antibodies, e.g., whichare specific binding partners for the molecule of interest.

Yet another immune component which may be identified and isolatedfollowing the invention is a cytolytic T cell ("CTL" hereafter),specific for complexes of peptides derived from the identified moleculeand MHC molecules to which these peptides bind, forming a complex. It isfairly well accepted that a CTL response involves the identification ofcomplexes of MHC molecules and peptides, generally about 8-12 aminoacids in length, but most preferably 9 or 10 amino acids in length, by Tcell receptors ("TCRs") on the surface of circulating T cells. The TCRsreact by binding to these complexes, setting in motion as it were aseries of reactions including the proliferation of CTLs specific forthese complexes. One can produce and/or isolate such CTLs using themethod of the invention, plus further steps.

As is pointed out in the examples which follow as well as the disclosurein general, one can easily identify cDNA encoding an antigen ofinterest. Once this is identified, one uses the cDNA to transfect hostcells which either already present desired MHC molecules on theirsurface, or which have been transfected with DNA encoding these MHCmolecules. The cDNA for the molecule of interest is expressed, and themolecule is processed to antigenic peptides which are presented by MHCmolecules, such as HLA molecules. CTLs directed against the complexesare obtained from lymphocytes, such as autologous lymphocytes. Fromresponder cell populations, long-term CTL clones are then obtained bythe well known technique of limiting dilution. Once a positive CTLresponse is observed, the specific peptides presented to the CTLs areidentified using established methods for example, screening the specificof previously identified CTL clones. Alternatively, the more recentlydescribed method of studying the sequence of the molecule of interest toidentify potential MHC-binding motifs then analyzing these peptides,first for binding to the relevant MHC molecule and then, if positive forMHC-binding, for their ability to generate CTLs recognizing the peptideMHC complex. Of course the peptides can also be eluted from the cellsand sequenced, using well known techniques.

It will also be noted by the skilled artisan that one can correlate theexpression of the molecule of interest back to a particular host cell orcells which expressed it. In so doing, one can remove the cDNA whichexpressed the molecule of interest, sequence it, and so forth. Thisaspect of the method is another feature of the invention.

Specific embodiments of the invention will be seen in the examples whichfollow. FIG. 1 depicts the method generally.

EXAMPLE 1

For the establishment of a cDNA library from human tissue total RNA wasobtained from 0.5 g of a renal clear cell carcinoma and establishedaccording to the method of Chomzynski, J. Analyt. Biochem. 162: 156-159(1987), incorporated by reference. The mRNA was extracted from total RNAwith oligo-dT-cellulose. The synthesis of the first strand cDNA wasaccomplished by the method described by Gubler and Hoffmann, Gene 25:263 (1983) using RNase H and DNA polymerase I. For adaptation of thecDNA Klenow enzyme, adaptors with EcoRI restriction enzyme sites wereligated to the cDNA ends using T4 DNA ligase (Ferretti V and SgamerellaV, Nucl. Acids Res. 9: 3695 (1981)). Following restriction enzymaticdigestion with the enzyme XhoI, cDNA molecules of different length wereseparated using Sephacryl 400 and transfected into λZAPII phage vectors(Short et al., Nucleic Acids Res. 16: 7583 (1988)). The recombinantphage DNA was packed into phages after ligation with packing extractsand used for the transfection of E. coli bacteria. The titration of thelibrary resulted in 1.8×10⁶ recombinant primary clones. The total cDNAlibrary was transfected in E. coli and amplified. The titer of the cDNAlibrary after amplification was 10¹¹ plaque forming units per ml(pfu/ml). These transfected cells were used in experiments which follow.

EXAMPLE 2

In accordance with the invention as described, supra, identification ofimmunogenic material was achieved by using human sera which have beencompletely depleted of antibodies directed against antigens derived fromnative and lytic λ phage-transfected E. coli bacteria. To this end, theserum was "stripped" via absorption, as now described.

E. coli bacteria of the strain XL1-blue were cultured in 50 ml LB mediumovernight. After achieving an optical density of OD₆₀₀ =1.0, thebacteria were pelleted by centrifugation, resuspended in 5 ml phosphatebuffered saline (PBS), and sonificated by ultrasound. The bacteriallysate was bound onto a matrix of activated Sepharose, which was thenput into a column and used for the absorption of the human serum. Theserum was run over this column 10 times.

A culture of E. coli XL1 blue bacteria in the exponential growth phasewas pelleted by centrifugation, transfected in 0.01 M magnesium sulfatewith 10⁶ λZAPII phages without a recombinant insert and incubated in 5ml LB medium for four hours. The lysate of the transfected bacteria wasused in the same manner as the untransfected bacteria, with the humanserum described supra being passed through the column an addition tentimes.

To complete the depletion of the serum, interfering antibodies fromlytically transfected E. coli bacteria were cultured on agar plates andtheir proteins were blotted onto nitrocellulose membranes after 10 hoursof culture at 37° C. Following this, the serum which had beenpreabsorbed according to the above steps was transferred to the blottednitrocellulose membrane, and the absorption procedure was repeated fivetimes. The serum, which was processed in accordance with the invention,was totally depleted of antibodies directed against antigens derivedfrom E. coli and phages.

EXAMPLE 3

In this, a renal cancer-specific antigen was identified via thefollowing steps. Bacteria of the strain E. coli XL1 blue weretransfected with recombinant phages derived from the described cDNAlibrary and plated at a density of 4-5×10³ plaque forming units (pfu)per plate in LB-medium with isopropylthiogalactopyranoside ("IPTG").After 12 hours of incubation at 37° C., nitrocellulose membranes wereput on top of the cultures and culture plates were incubated for anotherfour hours. This was followed by incubation of the nitrocellulosemembrane for one hour in Tris-buffered saline (TBS) with 5% milk powder.After washing the nitrocellulose membranes three times in TBS, thestripped human serum secured following Example 2 was diluted 1:1000 inTBS/0.5% (w/v) milk powder and incubated overnight with gentle shaking.After the incubation with the nitrocellulose membrane the serum wasremoved and kept for additional testing. Following incubation withserum, the nitrocellulose membranes were washed three times in TBS, andincubated with a polyclonal alkaline phosphatase-conjugated goatanti-human IgG serum for one hour. Following this, the nitrocellulosemembranes were washed repeatedly with TBS/0. 01% (v/v Tween 20). Thereaction was developed using nitroblue tetrazolium chloride andbromochloro-indoyl-phosphate in TBS. The binding of human antibodies tothe expressed protein became visible by a blue ring-formed color depositon the nitro-cellulose membrane. The efficient preabsorption of theserum made it possible to develop the membrane at 37° C. over severalhours without compromising the quality of the test because of backgroundreactivity caused by antibodies against E. coli and phage antigens.

Positive clones were localized on the agar plates, transferred intotransfection buffer, and used for a second round of transfection andsubcloning. A total of 1.8×10⁶ recombinant clones were subjected toscreening and five different positive-reacting clones were identified.

EXAMPLE 4

Positive clones secured following Example 3, i.e., those which had boundantibodies derived from the processed human serum, were subcloned tomonoclonality by repeated rounds of transfection and testing ofreactivity with the processed human serum. P-bluescript phagemids withthe respective cDNA inserts were cloned by in vivo excision (Hay andShort, Strategies 5: 16-19, 1992) from the λZAPII phage vectors and usedfor the transfection of E. coli SOLR bacteria. Plasmids were isolatedfrom the bacteria after alkaline lysis with NaOH in a modification ofthe method of Birnboim and Doly, J. Nucl. Acids Res. 7: 1513 (1979). Therecombinant plasmid DNA was sequenced according to the classic method ofSanger (Proc. Natl. Acad. Sci. USA 74: 5463 (1977)) using M13-forwardand M13-reverse oligonucleotides. The DNA sequence obtained and theresulting amino acid sequence were checked for in nucleic acid andprotein data banks (Gene Bank, EMBL, Swiss Prot). The sequencing of thecDNA inserts was continued using internal oligonucleotides. Analysisshowed no homology with any sequences deposited in the data banks. Thefull length cDNA clone referred to as SK313, which had been cloned withthe RACE method (Frohman M A, Dush M K, Martin G R, Proc. Natl. AcadSci. USA 85: 8998 (1988)), had a carbonic anhydrase domain at the 5'end. The nucleic acid sequence of this molecule is presented in SEQ IDNO: 1. FIG. 2 shows a nitrocellulose membrane with a positive clone fromthese experiments.

EXAMPLE 5

As a follow up to these experiments, RNA was isolated from a spectrum ofmalignant and normal human tissues according to the method of Chomzynskiand Sacchi Analyt Biochem. 162: 156 (1987). After denaturation, thetotal isolated RNA was separated on an agarose gel containing 1%formaldehyde by electrophoresis (Goldberg, Proc. Natl. Acad. Sci. USA77: 5794 (1980)) and then blotted onto a nylon membrane according to aknown method (Seed, Nucl. Acids Res. 10: 1799 (1982)) Radiolabeled cDNAinserts of the identified clones were used for hybridization. Thehybridization was carried out according to a known method (Geoffrey &Berger, Enzymol. 152: 419 (1987)). The presence of the respective RNAwas demonstrated using autoradiography and X-ray films. The analysisdemonstrated that the mRNA of clone HOM-RCC-313 was overexpressed in 4out of 19 renal cell carcinomas compared to normal kidneys. Very weakexpression was found only in colonic mucosal tissue and in normalkidney. Expression in other tissues could not be demonstrated.

EXAMPLE 6

To determine the incidence of antibodies against antigens which areidentified in accordance with the invention, sera from healthyindividuals and tumor patients were analyzed. To this end, the sera wereprocessed as described, supra, and depleted of antibodies againstantigens derived from E. coli and phages. For the detection ofantigen-specific antibodies, phages derived from reactive clones weremixed with non-reactive phages derived from the same cDNA library at aratio of 1:10 and tested as described supra for reactivity withantibodies in the human test serum. The serum which had been used forthe identification of the antigen was used as a positive clone. Thenon-reactive phages served as a negative control. A serum sample waspositive for antigen reactive antibodies, if the expected percentage ofthe phage plaques showed a positive reaction. In the case of the renalcell carcinoma antigen represented by clone HOM-RCC-313, the analysis ofa spectrum of human sera showed that only sera from renal cell carcinomapatients contained reactive antibodies. Sera from healthy controls andpatients with other tumors did not contain such antibodies.

The cDNA for clone HOM-RCC-313 was excised from the plasmid DNA bydigestion with the restriction enzyme EcoRI, was separated by agarosegel electrophoresis, followed by extraction from the gel. This was thenused to create a vector which expresses a fusion protein with thebacterial protein anthranilate synthetase. A relevant fragment in theexact open reading frame was cloned into pATH plasmid vectors (Koerner,Hill, Myers, Tzagaloff, Meth. Enzymol. 194: 477 (1991). Induction ofprotein expression was obtained after transformation of the plasmidsinto E. coli of strain BL21 as described (Spindler, Rosser, Berk, J.Virol. 49: 132 (1984)). Expressed, fusion proteins were separated by SDSgel electrophoresis, excised from the gel, eluted and freeze dried.Rabbits were immunized by subcutaneous injection with 100 μg of thelyophilisate dissolved in Freund's adjuvant. Immunization was repeatedthree times at two-week intervals using incomplete Freund's adjuvant.The rabbit was bled and antiserum was obtained. The obtained antiserumwas depleted from antibodies reactive with E. coli and phages in themanner described supra and tested for reactivity against the renalcarcinoma antigen as described for the human serum. Reactivity wasdetected at dilutions of 1:>100,000.

EXAMPLE 7

The protocols set forth in the preceding examples were followed, usingbiopsied tissue taken from different subjects suffering from (i)malignant melanoma, (ii) astrocytoma, and (iii) Hodgkin's Disease. Table1, which follows, summarized the results, including those obtained withthe renal cancer study, set out in detail in Examples 1-6, supra.

                  TABLE 1                                                         ______________________________________                                        Antibody reactivity of autologous sera with recombinant                        clones derived from human tumor CDNA. cDNA libraries were                     screened with autologous patient serum. Positive clones were                  subcloned to monoclonality. Inserts from each clone were amplified            with plasmid primers and separated by agarose gel electrophoresis.            Southern blots were performed by cross hybridization with the                 respective inserts.                                                                           clones      positive                                                                            different                                    tumor tested clones inserts                                                 ______________________________________                                        malignant melanoma                                                                         1.0 × 10.sup.6                                                                      40      10                                             renal cell carcinoma 1.8 × 10.sup.6  7 5                                astrocytoma 1.2 × 10.sup.6 49 5                                         Hodgkin's disease 1.0 × 10.sup.6 14 4                                 ______________________________________                                    

Following the analysis of the different inserts observed, the melanomacells expressed the known tumor rejection antigen precursor MAGE-1 (seevan der Bruggen et al., Science 254: 1643-7 (1991), incorporated byreference), as well as a new antigen. A portion of the cDNA sequence ofthis antigen is set forth in SEQ ID NO: 2.

When the astrocytoma study was completed, the observed insertcorresponded to the previously described Tegt gene (Old, Canc. Res. 41:361-375 (1981), incorporated by reference).

When the Hodgkin's Disease study was completed, a previously unknownantigen was isolated, and cDNA encoding it was identified in thelibrary, using standard methods. The antigen is a newly observed,lectin-like structure, a portion of the cDNA for which is set forth inSEQ ID NO: 3. Also observed were antibodies against restin, described byBilbe, et al, EMBO J 11: 2103-13 (1992). This is an intermediatefilament associated protein, expression of which has been shown to berestricted to Hodgkin and Reed-Sternberg cells, as well as culturedmonocytes.

EXAMPLE 8

A further study of occurrence of antibodies against the antigensdescribed in Examples 1-7 was carried out. Table 2 summarizes theseassays. In these studies, phages from positive clones were mixed withnon-reactive phage (ratio:1:10), and then used to transfect bacteria (e.coli). Dilutions of patient sera (1:200), were used, in an enzyme linkedimmunosorbent assay (ELISA), as described supra. "HOM-MEL-40" refers tothe new melanoma antigen (SEQ ID NO: 2), while "HOM-MEL-55" refers toMAGE-1 (van der Bruggen et al., supra). "HOM-RCC 3.1.3" is the renalcancer antigen of SEQ ID NO: 1. "HOM-GLO-30.2.1" refers to thepreviously identified astrocytoma associated antigen, "HOM-HD-21" refersto the new, lectin-like antigen of SEQ ID NO: 3, and "HOM-HD-397" is thepreviously identified restin antigen.

                                      TABLE 2                                     __________________________________________________________________________    Humoral immune responses against human tumor antigens. Phages from            positive clones were mixed with nonreactive phages of the                       cDNA-library at a ratio of 1:10 and used to transfect bacteria. IgG         antibodies to the clones were detected with an                                  enzyme-linked assay using sera. n.t.                                        antigen          HOM-MEL-55                                                                           HOM-RCC-3.1.3*                                                                         HOM-GLIO-30.2.1                                                                        HOM-HD-21*                                                                           HOM-HD-397                     identity/homology HOM-MEL-40* MAGE-1 CAH-like tegt lectin-like rectin       __________________________________________________________________________    melanoma patients                                                                      2/11    4/11   n.t.     n.t.     n.t.   n.t.                           recal cancer patients 0/8  0/8  2/14 0/7  0/7 5/7                             astrocytoma patients 0/10 0/10 0/11 2/13  0/11  7/11                          Hodgkin's patients 0/10 0/10 0/17 0/17 10/18 14/17                            healthly controls 0/12 0/12 0/15 0/20  0/17 12/17                           __________________________________________________________________________

The fact that antibodies against the tumor antigens, excepting onlyrestin, were detected, albeit at varying rates, only in the sera ofpatients diseased with the same type of tumor suggests that tumor growthis essential for the development of a humoral response against tumorantigens.

The reason for the presence of restin in healthy controls is not clear.One may speculate that tolerance against respective antigens might becircumvented, because the antigen may have similar sequences to anotherantigen, the donor may have premalignant cells, or the antigen may beactivated in normal cells under non-malignant conditions, such as viralinfections, or other inflammatory processes.

EXAMPLE 9

In order to determine the expression pattern of the newly identifiedantigens described herein, Northern blot analysis was carried out, usinga variety of human tissues.

RNA was extracted from tissue samples (tumor and normal) using the wellknown guanidium isothiocyanate/phenol/chloroform method of Chomzynski,et al., supra. The RNA integrity was checked via electrophoresis informalin/MOPS gels. Then, gels containing 40 ug of RNA per lane wereblotted onto nylon membranes. These Northern blots were then probed withthe cDNA of SEQ ID NO: 1, 2 or 3. Hybridization was with ³² P labelledprobes at 42° C., with formamide. The filters were washed at 65° C., at1×SSC, 0.2% SDS, and exposed for 16 hours. These are "stringentconditions" as defined hereafter. After exposure, filters were strippedand rehybridized with GAPDH.

Table 3 summarizes these results.

                                      TABLE 3                                     __________________________________________________________________________    Expression pattern of tumor antigens in various tissues (selection).          Northern-slot analysis was                                                      performed with RNA samples from tumor and normal human tissues matched      by hybridization                                                                with GAPDH. Expression ratios were calculated after densitometric           analysis of autoradiographs.                                                    The signal obtained with the normal coutnerpart of the diseased tissue      was set                                                                         to 1. n.t. = not tested;                                                    antigen         HOM-RCC-3.1.3*                                                                         HOM-GLIO-30.2.1                                                                        HOM-HD-21*                                    identity/homology HOM-MEL-40* CAH-like Tegt Lectin-like                     __________________________________________________________________________    kidney  -       1        1.5      --                                            brain - n.t. 1 n.t.                                                           tossil - -- 1 1                                                               stomach - -- 1.5 --                                                           colon neucosa - 0.2 1.5 --                                                    breast - -- 1.0 --                                                            renal cancer - >5 in 4/19 cases n.t. --                                        - ≦1 in 15/19 cases                                                   Hodgkins's tissue n.t. -- n.t. >10                                            astrocytoma n.t. n.t. >5 in 8/12 --                                              1 in 4/12                                                                  melanoma ++ -- n.t. --                                                      __________________________________________________________________________

As will be seen the new melanoma associated antigen is stronglyexpressed in melanoma, but not other tissues. Carbonic-anhydrase-likeantigen was strongly expressed in about 20% of renal cell carcinomas,and only weakly in normal renal tissue. Tegt was overexpressed on 8/12astrocytoma tissues compared to normal brain tissue. The mRNA for thelectin like molecule associated with Hodgkin's disease was increasedabout ten fold in diseased tonsils as compared to normal tonsils,suggesting that overexpression may be a frequent characteristic ofproteins which elicit autologous B cell responses.

EXAMPLE 10

Further studies were carried out on the HOM-MEL-40 sequence. Usingstandard genetic analysis techniques, the 5' region of the mRNA forHOM-MEL 40 was shown to have a tyrosine kinase binding domain. Thissuggests that HOM-MEL-40 may function as a receptor. The 3' portion ofthe RNA is identical with an RNA molecule for "SSX," a molecule known tobe involved in the SYT-SSX translocation in synovial tumors.

EXAMPLE 11

Additional experiments were also carried out to study HOM-MEL 40.Standard Northern blotting showed that, with the exception of testis,HOM-MEL 40 was not expressed in normal tissues. In contrast, it wasexpressed in 50% of melanomas, 75% of thyroid cancers, 25% of prostatecancers, 20% of gastric cancers, 16% of colorectal cancers, and 12% ofthe lung cancers tested.

Additional Western blotting work was carried out, showing thatantibodies against HOM-MEL 40 were present in 10 of 89 melanoma patientstested, but only 3 out of 49 healthy male subjects.

In yet further studies, it was observed that HLA-A2 positive tumor cellspresented a nonamer derived from HOM-MEL. This suggests that HOM-MEL 40specific vaccines, useful in inducing CTLs, are possible.

As the foregoing shows, the invention relates to a method fordetermining or isolating an immunoreactive substance. "Immunoreactivesubstance" as used herein refers to any material which provokes someform of immune response in the subject which produces it. This responsemay be based upon either a B cell or a T cell response. Suchimmunoreactive substances include proteins, peptides, glycoproteins,lipoproteins, peptide containing complexes (e.g., MHC/peptidecomplexes), antibodies, and so forth. To determine such substances, acDNA library is prepared from cells of a subject, using well known,standard methods. The cDNA is then inserted into an appropriate vector,such as a eukaryotic cell specific virus or a phage (i.e., a bacterialvirus), to form a transfecting/transforming library, which is thenincorporated into a host cell. The host cells are treated so that theyexpress the library component (cloned cDNA) they receive. The host cellsare then lysed, so that the expressed material is available for furthertreatment.

The lysed material is then contacted with a "stripped" sample believedto contain an immunogenic binding partner for the immunoreactivesubstance. "Immunogenic binding partner" as used herein refers to anyimmune system associated material which binds to the target, i.e., theimmunoreactive substance. Such binding partners include, but are notlimited to, antibodies, T cells, cytokines, ligands, receptors, and soforth, as well as truncated portions of these molecules, complementarynucleic acid molecules, and so forth. Note that for some of thesecomponents, such as T cells, further steps including those recitedherein are required.

The stripped sample, as indicated supra, has been treated by contactwith both (i) non-transfected or transformed host cells, and (ii) hostcells transfected or transformed with vectors which do not contain thepertinent cDNA.

The stripped sample is useful for identifying binding partners for theexpressed material because many of the immune components which wouldotherwise interfere with the specific immunological reaction desiredhave been removed via the absorption steps described herein.

The identification of the expressed material may be followed byisolation of the cDNA encoding it. One can punch holes through amembrane, such as a nitrocellulose membrane placed on top of Petridishes containing colonies of host cells, then use an immune reaction togive position on the solid phase. Each colony is based upon limited cDNAtransfection, thereby facilitating isolation and identification ofrelevant cDNA.

The invention also relates to the isolated nucleic acid molecules of SEQID NO: 1, 2 or 3, which encode for molecules which are associated withparticular conditions. In addition to their role as coding materials,these molecules can also be used as probes to identify cells expressingthe relevant antigens, as it has been shown that these cDNA molecules(SEQ ID NO: 1, 2 and 3) are based upon mRNA which translated to theantigen.

Also a part of the invention are isolated nucleic acid molecules, thecomplementary sequences of which hybridize to one of SEQ ID NO: 1, 2 or3, and which encode a protein equivalent to those encoded by SEQ ID NO:1, 2 or 3. "Stringent conditions" as used herein, refers to conditionsat least as stringent as hybridization at 50 μl/cm² of 3.5×SSC,1×Denhardt's solution, 25 mM sodium phosphate buffer (pH 7.0), using a³² P-labelled probe, for 18 hours at 65° C., followed by four washes(one hour, each wash, at 65° C., 2×SSC, 0.1% SDS), and a final wash for30 minutes at 1.0×SSC 0.2% SDS. The final wash can be changed to 0.5×SSCto 0.2×SSC, or even 0.1×SSC, and SDS can be lowered to 0.1% to increasestringency, if desired.

Other features of the invention will be clear to the skilled artisan andneed not be reiterated here.

The terms and expressions which have been employed are used as terms ofdescription and not of limitation, and there is no intention in the useof such terms and expressions of excluding any equivalents of thefeatures shown and described or portions thereof, it being recognizedthat various modifications are possible within the scope of theinvention.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES:  4                                          - -  - - (2) INFORMATION FOR SEQ ID NO:1:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 2679 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double-s - #tranded                                         (D) TOPOLOGY:  linear                                                - -    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                - - CGCGAAGATG CCCCGGCGCA GCCTGCACGC GGCGGCCGTG CTCCTGCTGG  - #                  50                                                                        - - TGATCTTAAA GGAACAGCCT TCCAGCCCGG CCCCAGTGAA CGGTTCCAAG  - #                 100                                                                         - - TGGACTTATT TTGGTCCTGA TGGGGAGAAT AGCTGGTCCA AGAAGTACCC  - #                 150                                                                         - - GTCGTGTGGG GGCCTGCTGC AGTCCCCCAT AGACCTGCAC AGTGACATCC  - #                 200                                                                         - - TCCAGTATGA CGCCAGCCTC ACGCCCCTCG AGTTCCAAGG CTACAATCTG  - #                 250                                                                         - - TCTGCCAACA AGCAGTTTCT CCTGACCAAC AATGGCCATT CAGTGAAGCT  - #                 300                                                                         - - GAACCTGCCC TCGGACATGC ACATCCAGGG CCTCCAGTCT CGCTACAGTG  - #                 350                                                                         - - CCACGCAGCT GCACCTGCAC TGGGGGAACC CGAATGACCC GCACGGCTCT  - #                 400                                                                         - - GAGCATACCG TCAGCGGACA GCACTTCTCC GCCGAGCTGC ACATTGTCCA  - #                 450                                                                         - - TTATAACTCA GACCTTTATC CTGACGACAG NACTGCCAGC AACAAGTCAG  - #                 500                                                                         - - AAGACCTCGC TGTCCTGGGT GCTCTCATTG AGATGGGCTC CTTCAATCCG  - #                 550                                                                         - - TCCTATGACA AGATCTTCAG TCACCTTCAA CATGTAAAGT ACAAAGGCCA  - #                 600                                                                         - - GGAAGCATTC GTCCCGGGAT TCAACATTGA AGAGCTGCTT CCGGAGAGGA  - #                 650                                                                         - - CCGCTGAATA TTACCGCTAC CGGGGGTCCC TGATCACACC CCCTTGCAAC  - #                 700                                                                         - - CCCACTGTGC TCTGGACAGT TTTCCGAAAC CCCGTGCAAA TTTCCCAGGA  - #                 750                                                                         - - GCAGCTGCTG GCTTTGGAGA CAGCCCTGTA CTGCACACAC ATGGACGACC  - #                 800                                                                         - - CTTCCCCCAG AGAAATGATC AACAACTTCC GGCAGGTCCA GAAGTTCGAT  - #                 850                                                                         - - GAGAGGCTGG TATACACCTC CTTCTCCCAA GTGCAAGTCT GTACTGCGGC  - #                 900                                                                         - - AGGACTGAGT CTGGGCATCA TCCTCTCACT GGCCCTGGCT GGCATTCTTG  - #                 950                                                                         - - GCATCTGTAT TGTGGTGGTG GTGTCCATTT GGCTTTTCAG AAGGAAGAGT  - #                1000                                                                         - - ATCAAAAAAG GTGATAACAA GGGAGTCATT TACAAGCCAG CCACCAAGAT  - #                1050                                                                         - - GGAGACTGAG GCCCACGCTT GAGGTCCCCG GAGCTCCCGG GCACATCCAG  - #                1100                                                                         - - GAAGGACCTT GCTTTGGACC CTACACACTT CGGCTCTCTG GACACTTGCG  - #                1150                                                                         - - ACACCTCAAG GTGTTCTCTG TAGCTCAATC TGCAAACATG CCAGGCCTCA  - #                1200                                                                         - - GGGATCCTCT GCTGGGTGCC TCCTTGTCTT GGGACCATGG NCACCCCAGA  - #                1250                                                                         - - GCCATCCGAT CGATGGATGG GATGCACTCT CAGACCAAGC AGCAGGAATT  - #                1300                                                                         - - CAAAGCTGCT TGCTGTAATT GTGTGAGATT GTGAAGTGGT CTGAATTCTG  - #                1350                                                                         - - GAATCACAAA CCAACCATGC TGGTGGGCCA TTAATGGTTG GAAAACACTT  - #                1400                                                                         - - CCATCCGGGG CTTTGCCAGA GCGTGCTTTC AAGTGTCCTG GAAATTCTGC  - #                1450                                                                         - - TGCTTCTCCA AGCTTTCAGA CAAGAATGTG CACTCTCTGC TTAGGTTTTG  - #                1500                                                                         - - CTTGGGAAAC TCAACTTCTT TCCTCTGGAG ACGGGACATC TCCCTCTGAT  - #                1550                                                                         - - TTCCTTCTGC TATGCAAAAC CTTTAATCTG CACCTTACAN ACTCGGGGAC  - #                1600                                                                         - - AAATGGGGAC AGGAAGGATC AAGTTGTAGA GNAGAAAAAG AAAACAAGAG  - #                1650                                                                         - - ATATACATTG TGATATATAT TAGGGACACT TTCACAGTCC TGTCCTCTGG  - #                1700                                                                         - - ATCACAGACA CTGCACAGAC CTTAGGGAAA TGGCAGGTTC AAAGTTCCAC  - #                1750                                                                         - - TTCTTGGTGG GGATGAGAAG GGAGAGAGAG CTAGAGGGAC AAAGAGAATG  - #                1800                                                                         - - AGAAGACATG GATGATCTGG GAGAGTCTCA CTTCGGAATC AGAATTGGAA  - #                1850                                                                         - - TCACATTCTG TTTATCAAGC CATAATGTAA GGACAGAATA ATACAATAAT  - #                1900                                                                         - - AAGTCCAAAT CCAACCTCCT GTCAGTGGAA CAGTTATGTT TTATACTCTA  - #                1950                                                                         - - CAGATTTTAC AAATANATGA GGCTNGTTCC TTGAAAANTG TGTTGNNTTG  - #                2000                                                                         - - CTGTNGTCCN NTGGAGGAGA CATGAGTTCC GAGATGACCA ACTCNNGCNT  - #                2050                                                                         - - TGNATNCTNG GAGGNAATAN GGCAGAACCA AAATGACTGT AGAACTTATT  - #                2100                                                                         - - CTCTGTAGGC CAAATTTCAT TTCAGCCACT TCTGCAGGAT CCTACTGCCA  - #                2150                                                                         - - ACCTGGAATG GAGACTTTTA TCTACTTCTC TCTCTCTGAA GATGTCAAAT  - #                2200                                                                         - - CGTGGTTTAG ATCAAATATA TTTCAAGCTA TAAAAGCAGG AGGTTATCTG  - #                2250                                                                         - - TGCAGGGGGC TGGCATCATG TATTTAGGGG CAAGTAATAA TGGAATGCTA  - #                2300                                                                         - - CTAAGATACT CCATATTCTT CCCCGAATCA CACAGACAGT TTCTGACAGG  - #                2350                                                                         - - CGCAACTCCT CCATTTTCCT CCCGCAGGTG AGAACCCTGT GGAGATGAGT  - #                2400                                                                         - - CAGTGCCATG ACTGAGAAGG AACCGACCCC TAGTTGAGAG CACCTTGCAG  - #                2450                                                                         - - TTCCCCGAGA ACTTTCTGAT TGCACAGTCT CATTTTGACA GCATGAAATG  - #                2500                                                                         - - TCCTCTTGAA GCATAGCTTT TTAAATATCT TTTTCCTTCT ACTCCTCCCT  - #                2550                                                                         - - CTGACTCTAG GAATTCTCTC TTCTGGAATC GCTTGAACCC AGGAGGCGGA  - #                2600                                                                         - - GGTTGCAGTA AGCCAAGGTC ATGCCACTGC ACTCTAGCCT GGGTGACAGA  - #                2650                                                                         - - GCGAGACTCC ATCTCAAAAA AAAAAAAAA         - #                  - #              2679                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:2:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 931 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double-s - #tranded                                         (D) TOPOLOGY:  linear                                                - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                               - - ACTTTCTCTC TCTTTCGATT CTTCCATACT CAGAGTACGC ACGGTCTGAT  - #                  50                                                                         - - TTTCTCTTTG GATTCTTCCA AAATCAGAGT CAGACTGCTC CCGGTGCCAT  - #                 100                                                                         - - GAACGGAGAC GACGCCTTTG CAAGGAGACC CACGGTTGGT GCTCAAATAC  - #                 150                                                                         - - CAGAGAAGAT CCAAAAGGCC TTCGATGATA TTGCCAAATA CTTCTCTAAG  - #                 200                                                                         - - GAAGAGTGGG AAAAGATGAA AGCCTCGGAG AAAATCTTCT ATGTGTATAT  - #                 250                                                                         - - GAAGAGAAAG TATGAGGCTA TGACTAAACT AGGTTTCAAG GCCACCCTCC  - #                 300                                                                         - - CACCTTTCAT GTGTAATAAA CGGGCCGAAG ACTTCCAGGG GAATGATTTG  - #                 350                                                                         - - GATAATGACC CTAACCGTGG GAATCAGGTT GAACGTCCTC AGATGACTTT  - #                 400                                                                         - - CGGCAGGCTC CAGGGAATCT CCCCGAAGAT CATGCCCAAG AAGCCAGCAG  - #                 450                                                                         - - AGGAAGGAAA TGATTCGGAG GAAGTGCCAG AAGCATCTGG CCCACAAAAT  - #                 500                                                                         - - GATGGGAAAG AGCTGTGCCC CCCGGGAAAA CCAACTACCT CTGAGAAGAT  - #                 550                                                                         - - TCACGAGAGA TCTGGACCCA AAAGGGGGGA ACATGCCTGG ACCCACAGAC  - #                 600                                                                         - - TGCGTGAGAG AAAACAGCTG GTGATTTATG AAGAGATCAG CGACCCTGAG  - #                 650                                                                         - - GAAGATGACG AGTAACTCCC CTCAGGGATA CGACACATGC CCATGATGAG  - #                 700                                                                         - - AAGCAGAACG TGGTGACCTT TCACGAACAT GGGCATGGCT GCGGACCCCT  - #                 750                                                                         - - CGTCATCAGG TGCATAGCAA GTGAAAGCAA GTGTTCACAA CAGTGAAAAG  - #                 800                                                                         - - TTGAGCGTCA TTTTTCTTAG TGTGCCAAGA GTTCGATGTT AGCGTTTACG  - #                 850                                                                         - - TTGTATTTTC TTACACTGTG TCATTCTGTT AGATACTAAC ATTTCATTGA  - #                 900                                                                         - - TGACGAAGAC ATACTTAATC GATATTTGGT T        - #                  - #             931                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:3:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1692 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double-s - #tranded                                         (D) TOPOLOGY:  linear                                                - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                               - - GATCCCCCGG GCTGCAGGAA TTCGGCACGA GCAAAGGACT TCCTAGTGGG  - #                  50                                                                         - - TGTGAAAGGC AGCGGTGGCC ACAGAGGCGG CGGAGAGATG GCCTTCAGCG  - #                 100                                                                         - - GTTCCCAGGC TCCCTACCTG AGTCCAGCTG TCCCCTTTTC TGGGACTATT  - #                 150                                                                         - - CAAGGAGGTC TCCAGGACGG ACTTCAGATC ACTGTCAATG GGACCGTTCT  - #                 200                                                                         - - CAGCTCCAGT GGAACCAGGT TTGCTGTGAA CTTTCAGACT GGCTTCAGTG  - #                 250                                                                         - - GAAATGACAT TGCCTTCCAC TTCAACCCTC GGTTTGAAGA TGGAGGGTAC  - #                 300                                                                         - - TTGGTGTCCA ACACGAGGCA GAACGGAAGC TGGGGGCCCG AGGAGAGGAA  - #                 350                                                                         - - GACACACATG CCTTNCCAGA AGGGGATGCC CTTTGACCTC TGCTTCCTGG  - #                 400                                                                         - - TGCAGAGCTC AGATTTCAAG GTGATGGTGA ACGGGATCCT CTTCGTGCAG  - #                 450                                                                         - - TACTTCACAT CTCGTCATGC CCTGTCCACC GTTGTGGACA CCATCTCCGT  - #                 500                                                                         - - CAATGGCTCT GTGCAGCTGT CCTACATCAG CTTCCAGCCT CCCGGCGTGT  - #                 550                                                                         - - GGCCTGCCAA CCCGGCTCCC ATTACCCAGA CAGNNNTCAT CCACACAGTN  - #                 600                                                                         - - GCAGAGCGCC CNCTGGACAG ATGTCTCTAC TCCCGCCATC CCACCTATGA  - #                 650                                                                         - - TGTACCCCCA CCCCGCCTAT CCGATGCCTT TCATCACCAC CATTCTGGGA  - #                 700                                                                         - - GGGCTGTACC CATCCAAGTC CATCCTCCTG TCAGGCACTG TNCTGCCCAG  - #                 750                                                                         - - TGCTCANGAG GTTCCACATC NAACCTGTGC NCTGGGAACC ACATCGCCTT  - #                 800                                                                         - - CCACCTGAAC CCCCGTTTTG ATGAGAATGC TGTGGTCCGC AACACCCAGA  - #                 850                                                                         - - TCGACAACTC CTGGGGGTCT CAGGAGCGAA GTCTGCCCCG AAAAATGCCC  - #                 900                                                                         - - TTCGTCCGTG GCCAGAGCTT CTCAGTGTGG ATCTTGTGTG AAGCTCACTG  - #                 950                                                                         - - CCTCAAGGTG GCCGTGGATG GTCAGCACCT GTTTGAATAC AACCATCGCC  - #                1000                                                                         - - TGAGGAACCT GCCCACCATC AACAGACTGG AAGTGGGGGG CGACATCCAG  - #                1050                                                                         - - CTGACCATGT GCAGACATAG GCGGCTTCCT GGCCCTGGGG CCGGGGGCTG  - #                1100                                                                         - - GGGTGTGGGG CAGTCTGGGT CCTCTCATCA TCCCCACTTC CCAGGCCCAG  - #                1150                                                                         - - CCTTTCCAAC CCTGCCTGGG ATCTGGGCTT TAATGCAGAG GCCATGTCCT  - #                1200                                                                         - - TGTCTGGTCC TGCTTCTGGC TACAGCCACC CTGGAACGGA GAAGGCAGCT  - #                1250                                                                         - - GACGGGGATT GCCTCCTCAG CCGCAGCAGC ACCTGGGGCT CCAGCTGCTG  - #                1300                                                                         - - GAATCCTACC ATCCCAGGAY GCAGGCACAG CCAGGGAGAG GGGAGGNGTG  - #                1350                                                                         - - GGCAGTGAAG ATGAAGCCCC ATGCTCAGTC CCCTCCCATC CCCCACGCAG  - #                1400                                                                         - - CTCCACCCCA GTCCCAAGCC ACCAGCTGTC TGCTCCTGGT GGGAGGTGGC  - #                1450                                                                         - - CTCCTCAGCN CCTCCTCTCT GACCTTTAAC CTNACTCTCA CCTTGCACCG  - #                1500                                                                         - - TGCACCAACC CTTCACCCCT CCTGGAAAGC AGGCCTGATG GCTTCCCACT  - #                1550                                                                         - - GGCCTCCACC ACCTGACCAG AGTGTTCTCT TCAGAGGACT GGCTCCTTTC  - #                1600                                                                         - - CCAGTGTCCT TAAAATAAAG AAATGAAAAT NCTTGTTGGC AAAAAAAAAA  - #                1650                                                                         - - AAAAAAAAAC TCGAGGGGCN NCCCNGTACC CAATTCGCCC TA    - #                      - #1692                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:4:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1190 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double-s - #tranded                                         (D) TOPOLOGY:  linear                                                - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                               - - ATCTGCAGAA TTCGGCTTCG ATCTAGAACT AGTGGATCCC CCGGGCTGCA  - #                  50                                                                         - - GGAATTCGGC ACGAGCGGTT CCAAGTGGAC TTATTTTGGT CCTGATGGGG  - #                 100                                                                         - - AGAATAGCTG GTCCAAGAAG TACCCGTCGT GTGGGGGCCT GCTGCAGTCC  - #                 150                                                                         - - CCCATAGACC TGCACAGTGA CATCCTCCAG TATGACGCCA GCCTCACGCC  - #                 200                                                                         - - CCTCGAGTTC CAAGGCTACA ATCTGTCTGC CAACAAGCAG TTTCTCCTGA  - #                 250                                                                         - - CCAACAATGG CCATTCAGTG AAGCTGAACC TGCCCTCGGA CATGCACATC  - #                 300                                                                         - - CAGGGCCTCC AGTCTCGCTA CAGTGCCACG CAGCTGCACC TGCACTGGGG  - #                 350                                                                         - - GAACCCGAAT GACCCGCACG GCTCTGAGCA TACCGTCAGC GGACAGCACT  - #                 400                                                                         - - TCTCCGCCGA GCTGCACATT GTCCATTATA ACTCAGACCT TTATCCTGAC  - #                 450                                                                         - - GACAGNACTG CCAGCAACAA GTCAGAAGAC CTCGCTGTCC TGGGTGCTCT  - #                 500                                                                         - - CATTGAGATG GGCTCCTTCA ATCCGTCCTA TGACAAGATC TTCAGTCACC  - #                 550                                                                         - - TTCAACATGT AAAGTACAAA GGCCAGGAAG CATTCGTCCC GGGATTCAAC  - #                 600                                                                         - - ATTGAAGAGC TGCTTCCGGA GAGGACCGCT GAATATTACC GCTACCGGGG  - #                 650                                                                         - - GTCCCTGATC ACACCCCCTT GCAACCCCAC TGTGCTCTGG ACAGTTTTCC  - #                 700                                                                         - - GAAACCCCGT GCAAATTTCC CAGGAGCAGC TGCTGGCTTT GGAGACAGCC  - #                 750                                                                         - - CTGTACTGCA CACACATGGA CGACCCTTCC CCCAGAGAAA TGATCAACAA  - #                 800                                                                         - - CTNCCGGCAG GTCCAGAAGT TCGNTGAGAG GCTGGTATAC ACCTCCTTCT  - #                 850                                                                         - - CNCAAGTGCA AGTCTGTACT GCGGCAGGAC TGAGTCTGGG CATCATCCTC  - #                 900                                                                         - - TCACTGGCCC TGGCTGGCAT TCTTGGCATC TGTATTGTGG TGGTGGTGTC  - #                 950                                                                         - - CATTTGGCTT TTCAGAAGGA AGAGTANCCC CNAAAGGTGA TAACAAGGGA  - #                1000                                                                         - - GTCATTTACA AGCCANCCAC CAAGATGGAG ACTGAGGCCC ACGCTTGAGG  - #                1050                                                                         - - TCCCCGGAGC TCCCGGGCAC ATCCAGGAAG GACCTTGCTT TTGGACCCTA  - #                1100                                                                         - - CACACTTCGG CTCTCTGGAC ACTTGCGACA CCTCAAGGTG TTCTCTGTAG  - #                1150                                                                         - - CTCAATCTGC AAACATGCCA GGCCTCAGGG ATCCTCTGCT     - #                      - #  1190                                                                    __________________________________________________________________________

I claim:
 1. Isolated nucleic acid molecule which encodes a proteinassociated with melanoma, said protein having an amino acid sequenceidentical to the amino acid sequence encoded by SEQ ID NO:
 2. 2. Theisolated nucleic acid molecule of claim 1, having a nucleotide sequenceconsisting of the nucleotide sequence set forth in SEQ ID NO:
 2. 3.Expression vector comprising the isolated nucleic acid molecule of claim1, operably linked to a promoter.
 4. Expression vector comprising theisolated nucleic acid molecules of claim 2, operably linked to apromoter.
 5. Cell line transformed or transfected with the isolatednucleic acid molecule of claim
 1. 6. Cell line transformed ortransfected with the isolated nucleic acid molecule of claim
 2. 7. Cellline transformed or transfected with the expression vector of claim 3.8. Cell line transformed or transfected with the expression vector ofclaim 4.